The human and animal trypanosomiases have major medical and veterinary consequences. Infections of Trypanosoma ssp. and Leishmania ssp. cause mortality as well as debilitating disease that curtail human productivity. Trypano-tolerant cattle, which are the only cattle that can survive in sub-saharan Africa, are inefficient meat and milk producers and thus contribute to the food deficit in Africa. Several unusual characteristics of trypansomatidae relate to mRNA production. Mature mRNAs of trypanosomatids are formed by a process that probably involves dissection of large polycistronic primary mRNA transcripts by splicing of a 39 nucleotide mini-exon (MX) in trans to the 5' end of the mature mRNA. The 39 nucleotide MX initially comprises the 5' end of a short, non- polyadenylated RNA that is transcribed from a set of tandemly reiterated genes. Although the genes that encode the MX have been cloned, protein components responsible for the transcription of these genes remain obscure. Drug-sensitivity experiments and DNA sequence analyses have demonstrated that although there appear to be the classical three types of RNA polymerases in trypanosomes, the MX gene contains components reminiscent of both polymerase II and III-directed genes. Although there is a vast literature on gene regulation in several prokaryotic and eukaryotic organisms, virtually nothing is known about RNA transcription in trypanosomatids. The uniqueness of the MX gene structure and MX RNA function makes this gene an appropriate point in which to start this analysis. An understanding of MX transcription will uncover proteins that are common to all trypanosomatids but distinct from proteins responsible for RNA synthesis in mammalian cells. We have used reverse genetic analyses of the MX genes in the model insect trypanosomatid Leptomonas seymouri to determine which sequences within the gene repeat are necessary for MX RNA synthesis. We have used this information to develop an assay for detecting specific protein-DNA interactions. We plan to use this assay for the biochemical purification of transcription factors that are components of the MX transcription initiation complex. Ultimately, we hope to uncover the cellular mechanisms responsible for MX transcription. This knowledge of the expression of a vital trypanosomatid gene will enable us to contribute to the rational drug design of anti-parasitic agents.